Methods for culturing undifferentiated cells using sustained release compositions

ABSTRACT

Methods for culturing undifferentiated mammalian cells, such as stem and progenitor cells, are provided. The methods involve incubating the cell in the presence of a sustained release composition containing at least one growth factor, wherein the sustained release composition continuously releases the growth factor(s), and wherein the presence of the sustained level of growth factor maintains the cell in an undifferentiated state.

RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/360,741, filed Jul. 1, 2010, which is hereinincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithvia EFS-Web as an ASCII compliant text file named “Sequence Listing.txt”that was created on Jun. 17, 2011, and has a size of 12,983 bytes. Thecontent of the aforementioned file named “Sequence Listing.txt” ishereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention provides improved methods for culturingundifferentiated mammalian cells, such as stem and progenitor cells. Themethods involve incubating the cell in the presence of a sustained levelof at least one growth factor. A sustained release composition thatcontinuously releases the growth factor is used to maintain sustainedlevels of the growth factor, and wherein the presence of the sustainedlevels of growth factor maintains the cell in an undifferentiated stateand increases the quantity and/or quality of cells produced.

BACKGROUND OF THE INVENTION

Stem cells are undifferentiated cells that possess two hallmarkproperties; self-renewal and the ability to differentiate into one ormore different cell lineages. The process of self-renewal involves theself-replication of a stem cell to allow for propagation and expansion,wherein the stem cell remains in an undifferentiated state. Progenitorcells are also undifferentiated cells that have the ability todifferentiate into one or more cell lineages, but have limited or noability to self renew. When maintained in culture, undifferentiatedcells, such as stem or progenitor cells, can undergo spontaneousdifferentiation, thereby losing the desired, undifferentiated cellphenotype. Thus, culture methods that minimize spontaneousdifferentiation in order to maintain the undifferentiated stem orprogenitor cell state are needed.

Keeping undifferentiated cells, such as, but not limited to, stem and/orprogenitor cells in an undifferentiated state is critical to their use,e.g., in industry and medicine, since a major scientific and therapeuticusefulness of these cells lies in their ability to expand intohomogenous populations that can further proliferate or differentiateinto mature cells as needed, e.g., for scientific study or to repairdamage to cells or tissues of a patient. Once they have spontaneouslydifferentiated in cell culture, the cells are less proliferative andless able to differentiate into different types of cells as needed. Ahomogenous culture of undifferentiated stem cells is therefore a highlysought after but unrealized goal of research scientists and industry.

Current methods for culturing undifferentiated cells (e.g., varioustypes of stem cells) attempt to minimize such spontaneousdifferentiation by delivering fibroblast growth factor 2 (FGF2) to thecell cultures daily, or, less frequently than every day, which is knownas “feeding”. FGF2 has been shown to promote self-renewal of stem cellsby inhibiting differentiation of the stem cell; however this inhibitionis incomplete, and the stem cell cultures tend to graduallydifferentiate, thereby diminishing usefulness of the stem cell culture.Furthermore, stem cells, such as ES cells, typically need to be grown onmouse embryonic fibroblast (MEF) feeder cells. This is a cumbersome stepthat is desirable to remove.

For human embryonic stem cells (hESC), as well as other undifferentiatedcell types, FGF2 is required for the maintenance of the undifferentiatedstate, and withdrawal of FGF2 from the culture conditions initiatesdifferentiation. [See, Amit, M., et al. (2000) Dev Biol. 2, 271-78;Itskovitz-Eldor, J., et al. (2000) Mol Med. 2, 88-95; Xu, C., et al.(2001) Nat Biotechnol. 10, 971-74; Xu et al., 2005; Xu, R. H., et al.(2005) 3, 164-65; Ding, V., et al. (2006) Biotechnol Lett. 7, 491-95;Levenstein, M.E., et al. (2006) Stem Cells 3, 568-74; Ludwig, T. E., etal. (2006) Nat Methods. 8, 637-46; Bendall, S. C., et al. Nature 448,1015-21]. FGF2 is also required to maintain neural stem cells (NSC) andneural progenitor cells in an undifferentiated state. [See, Temple S.(1989). Nature 340:471-473.; Vescovi, A. L., et al. (1993) Neuron 5,951-56; Kilpatrick, T. J., and Bartlett, P. F. (1995) J Neurosci. 5,3653-61; Temple, S., and Qian, X. (1995) Neuron 2, 249-52; Qian, X., etal. (1997) Neuron 1, 81-83; Ciccolini, F., and Svendsen, C. N. (1998) JNeurosci. 19, 7869-80; Vaccarino F M, et al. (1999) Curr Top Dev Biol.46 179-00; Raballo R, et al. J Neurosci. 13, 5012-23.] In traditionalmethods for culturing NSCs, growth factors such as FGF2 are onlyreplenished once every three days. However, NSCs cultured by thesemethods are reported to have high rates of spontaneous differentiation[Qian et al., 1997, supra].

Growth factors, such as FGF2, are understood to work directly on hESCs,NSCs, and other stem and progenitor cells, and/or, in some methods,indirectly by stimulating feeder cells in the cell culture to producethis and other growth factors [Bendall, et al., 2007, supra]. However,levels of FGF2 and other growth factors are unstable in these cellcultures, and must be frequently replaced. For example, the half-life ofFGF2 is less than 24 hours under conditions typically used to culturestem and progenitor cells. [McKinnon et al., 1990, Neuron. 1990November; 5 (5):603-14]. Consequently, standard methods for maintaininghESC cultures require feeding the cells every day with soluble FGF2and/or other growth factors in order to maintain effective amounts ofactive FGF2 polypeptide and/or those other growth factors [Fasano C A,et al. (2010) Cell Stem Cell 6, 336-47]. Despite that laborious,time-consuming process, however, daily feeding of FGF2 and/or othergrowth factors to stem and progenitor cells still results in (1)significant variation of growth factor levels, with very high levels ofFGF2 for the few hours immediately after feeding and very low FGF2levels present during the few hours prior to the next daily feeding, and(2) limited effectiveness, since hESCs still gradually differentiate,albeit at a slower rate than in the absence of daily feeding with FGF2.

Biodegradable “microspheres” and “millicylinders” prepared frombiocompatible polyesters of glycolic and lactic acids (“PLGA”) are knownfor delivering protein drugs to patients, and PLGA millicylindersencapsulated with recombinant human FGF2 (also known as “basicfibroblast growth factor” or “bFGF”) have been described by Zhu et al.(Nature Biotechnology (2000) 18:52-57) for such applications. Olaye etal. (European Cells and Materials (2008) 16 (Suppl. 3):86) teach that“PLGA microspheres have been extensively used for the sustained deliveryof growth factors for embryonic stem cell differentiation,” and reportthat PLGA microsphere-based scaffolds were successfully used to delivercertain growth factors—specifically Asc, Dex and TGF-β₁—fordifferentiation of murine embryonic stem cells into osteoblast andchondrocyte-like cells. PVA-based polymer coatings and hydrogenparticles for cell culturing have also been described, e.g., by Hemperlyet al., U.S. Patent Application Publication No. 2004/0209361 and Keithet al., U.S. Patent Application Publication No. 2004/0209360,respectively. These polymer coatings and particles promote celladhesion, and may also provide slow release of “bioaffecting molecules,”such as growth factors. Id. More recent publications emphasize a rolefor FGF2 in promoting cellular differentiation, and discuss hydrogels,microspheres and the like for tissue specific delivery of that growthfact, e.g., to promote tissue regeneration and wound healing. Forreview, see Yun et al., J. Tissue Eng. (Nov. 7, 2010) 2010:218142; seealso Macdonald et al., Biomacromolecules (Aug. 9, 2010) 11(8):2053-2059. Hence, the use of such “sustained release” preparationsin stem cell cultures has been limited to delivering growth factors forstem cell differentiation. The sustained release of growth factors,using such compositions or otherwise, for maintaining cells in anundifferentiated state is believed to be heretofore unknown. Hence,there remains a need in the art for improved methods of culturing stemcells and other undifferentiated cells, and for maintaining such cellsin an undifferentiated state.

SUMMARY OF THE INVENTION

As follows from the Background Section, there is a clear need forcompositions and methods for culturing undifferentiated cells, such asESCs, NSCs, and other types of undifferentiated cells (e.g. RPESCs,iPSCs, SCSCs, etc.) that reduces or eliminates their spontaneousdifferentiation. There is also a need in the art for compositions andmethods to reduce the time, labor and expense currently required forculturing undifferentiated cells. These and other problems which will beapparent to persons of ordinary skill in the art are at least partiallysolved by the present invention.

Thus, in one aspect, the present invention provides a method forculturing a mammalian stem or progenitor cell, wherein the methodcomprises incubating the stem or progenitor cell in the presence of astable concentration range of at least one growth factor over a periodof at least 1 day. In certain embodiments, the stable concentrationrange of growth factor is maintained by continuous release of the growthfactor using mechanical means. In other embodiments, the stableconcentration range of growth factor is maintained by a sustainedrelease composition, such as a PLGA microsphere containing the growthfactor.

In another aspect, the present invention provides a method for culturinga mammalian stem or progenitor cell, wherein the method comprisesincubating the stem or progenitor cell in the presence of a sustainedrelease composition containing at least one growth factor, wherein thesustained release composition releases the growth factor, and whereinthe presence of the growth factor maintains the cell in anundifferentiated state. In certain embodiments, the released growthfactor is maintained within a predetermined concentration range.

In another aspect, the present invention provides a method for culturinga stem or progenitor cell, wherein the method comprises incubating thestem or progenitor cell in the presence of a sustained releasecomposition comprising fibroblast growth factor 2 (FGF2), wherein thesustained release composition releases FGF2. In certain embodiments, thesustained release composition comprises FGF2 at a concentration of 0.5%(w/v) at the start of incubation with the stem or progenitor cell.Furthermore, the released FGF2 is maintained within a predeterminedconcentration range.

In yet another aspect, the present invention provides a method forculturing an undifferentiated mammalian cell, wherein the methodcomprises incubating the undifferentiated mammalian cell in the presenceof a sustained release composition containing at least one growthfactor, wherein the sustained release composition releases the growthfactor, wherein the released growth factor is maintained within apredetermined concentration range, and wherein the maintenance of thereleased growth factor within said concentration range maintains thecell in an undifferentiated state.

In any of the above methods for culturing the stem or progenitor cell orundifferentiated mammalian cell, the sustained release of the growthfactor may maintain the concentration of the growth factor in the cellculture in a stable concentration range of 80%-100%, 80%-95%, or 80%-90%of the starting concentration of growth factor.

In any of the above methods for culturing the stem or progenitor cell orundifferentiated mammalian cell, the sustained release composition mayrelease at least one growth factor over a period of over at least 1 day,at least 2 days, at least 3 days, at least 4 days, at least 5 days, atleast 6 days, or at least 7 days, or longer, e.g., 2 weeks, 3 weeks, 4weeks, 5 weeks, or longer.

In any of the above methods for culturing the stem or progenitor cell orundifferentiated mammalian cell, the cell may be maintained in anundifferentiated state for over at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, or atleast 7 days, or longer, e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, orlonger.

In any of the above methods for culturing the stem or progenitor cell orundifferentiated mammalian cell, the sustained release composition is apoly(DL-lactide-co-glycolide) (PLGA) microsphere. In certainembodiments, the concentration of the PLGA microsphere ranges from about5 to about 300 ng/ml.

In any of the above methods for culturing the stem or progenitor cell orundifferentiated mammalian cell, the sustained release composition mayfurther comprises one or more of heparin, dextran sulfate, Mg(OH)₂, apolyanion that complexes with growth factor, and/or EDTA. In certainembodiments, the sustained release composition further comprises 1.0%(w/v) heparin and/or 1.0% (w/v) dextran sulfate. In other embodiments,the ratio of heparin or dextran sulfate to FGF2 is about 2:1. In stillother embodiments, the sustained release composition further comprises3% (w/v) Mg(OH)₂. In yet other embodiments, the sustained releasecomposition further comprises 1 mM EDTA.

In any of the above methods for culturing the stem or progenitor cell orundifferentiated mammalian cell, the stem cell is selected from thegroup consisting of an embryonic stem cell, an induced-pluripotent stemcell, a neural stem cell, a retinal pigment epithelial stem cell, amesenschymal stem cell, a hematopoietic stem cell, an epiblast stem cellor a cancer stem cell. In certain embodiments, the stem cell is a humanembryonic stem cell or a neural progenitor cell.

In any of the above methods for culturing the stem or progenitor cell orundifferentiated mammalian cell, the sustained release compositionfurther comprises one or more additional growth factors. In certainembodiments, the one or more additional growth factors are selected fromthe group consisting of epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), sonic hedgehog (Shh), leukemia inhibitory factor(LIF) and a Wnt protein.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 contains graphs showing the percent (%) change in FGF2concentrations 5-6 hours after culturing hESCs on mouse embryonicfibroblasts (MEFs) (FIG. 1A) or on Matrigel (non-MEF) (FIG. 1B) incomplete hESC culture media containing 10 ng/ml FGF2 (“C′ media+FGF2”)or MEF conditioned media containing 10 ng/ml FGF2 (“MEF CM+FGF2”) mediarelative to the concentration at the start of culture.

FIG. 2 is a graph showing the percent (%) change in FGF2 concentrationafter 6 hours, on Day 1, Day 2, and Day 3 in the culture media of cellscultured with or without FGF2-containing PLGA microspheres(“FGF2-microspheres”) added on Day 0, following replacement of theculture media with fresh complete media containing 10 ng/ml FGF2.Percent (%) change is relative to the starting concentration of FGF2 onDay 0, immediately following replacement of the culture media and beforeaddition of microspheres (in the FGF2-microspheres group).

FIG. 3 is a graph showing the percent (%) (relative to soluble FGF2treated group) of SSEA-4 mRNA expression in human ESCs treated withsoluble FGF2, FGF2-containing PLGA microspheres (“FGF2-microspheres”) orempty microspheres, 5 days after treatment.

FIG. 4 is a graph showing the percent (relative to soluble FGF2 treatedgroup) of OCT4, Brachyury and SOX17 mRNA expression in human ESCstreated with soluble FGF2, FGF2-containing PLGA microspheres or emptymicrospheres (negative control), 5 days after treatment.

FIG. 5A is a graph showing the percent of cells that stained positivefor SSEA-1, SSEA-3 and SSEA-4 protein on Day 35 in cultures of WA-09hESCs that were fed every day, on Day 0 and then every third day for theduration of culture (“Every 3^(rd)”) or on Day 0 (“Once”) with 10 ng/mlFGF2, or in cultures of WA-09 hESCs that were fed with FGF2-containingPLGA microspheres on Day 0 (“Beads Once”) or on Day 0 and then everythird day for the duration of culture (“Beads Every 3^(rd)”) “*”indicates that p<0.05 compared to the “Every Day” group, for the sameprotein, and “#” indicates that p<0.05 compared to the “Every 3^(rd)”group, for the same protein.

FIG. 5B is a graph showing fold change in mRNA expression of NANOG onDay 14 and Day 21 in WA-09 hESCs that were fed every day, on Day 0 andthen every third day for the duration of culture (“Every 3^(rd)”) or onDay 0 (“Once”) with 10 ng/ml FGF2, or in cultures of WA-09 hESCs thatwere fed with FGF2-containing PLGA microspheres on Day 0 (“Beads Once”)or on Day 0 and then every third day for the duration of culture (“Beads3^(rd) Day”).

FIG. 6 is a graph showing the fold change in mRNA expression of Oct-4and Sox17 in WA-09 hESCs cultured for 5 days on Matrigel in the presenceof MEF-conditioned medium and either soluble FGF2 or FGF2-containingmicrospheres. In the control group, soluble FGF2 was added at aconcentration of 10 ng/ml every day (“Every Day”). FGF2-containing PLGAmicrospheres were added to the cell culture once on Day 0 (“Beads FedOnce”) or on Days 0 and 2 (“Beads Twice per Week”).

FIG. 7A is a graph showing the percent (%) of cells that stainedpositive for SSEA-3 or SSEA-4 protein on Day 7 in hESCs cultured withdaily feeding of FGF2 (“FGF2 daily”) or in hESCs plated in mediacontaining 10 ng/ml FGF2 and then not fed again (“No FGF2”). FIG. 7B isa graph showing the percent of cells in the “FGF2 daily” or “No FGF2”groups that stained positive for SSEA-3 protein following an additional7 days of culture in the following conditions: cells were fed every daywith 10 ng/ml FGF2 (“Every day”), or with FGF2 microspheres on Day 0(“Once”) or on Days 0 and 2 (“Beads Twice per Week”).

FIG. 8 is a graph showing the number of cells expressing β-tubulin orNestin in the indicated treatment groups, 7 days after treatment ofNSCs.

FIG. 9 is graph showing the number of cells expressing Nestin orβ-tubulin in the indicated treatment groups, 6 days after treatment ofNSCs with FGF2 according to the standard method (every 3rd day) or every8 hours (hrs).

FIG. 10 is a photograph of RPESCs following 5 days of culture in thepresence of FGF2-containing PLGA microspheres (32× magnification).

FIG. 11 contains photographs of iPSCs taken 9 days after the cells wereplated and cultured with daily addition of soluble FGF2 (upper panel) orwith the addition of FGF2-containing PLGA microspheres on Days 0 and 3(lower panel) (20× magnification).

DETAILED DESCRIPTION I. Definitions

As used herein, the term “stem cell” refers to a cell that retains theability to renew itself through mitotic cell division, and candifferentiate into a diverse range of specialized cell types. The term“stem cell” includes by way of non-limiting examples, neural stem cells(NSCs), embryonic stem cells (ESCs), induced pluripotent stem cells(iPSCs), hematopoietic stem cells (HSCs), cancer stem cells (CSCs),spinal cord stem cells (CSCs), mesenchymal stem cells (MSCs), retinalpigment epithelial stem cells (RPESCs), and epiblast stem cells. As usedherein, the term “embryonic stem cell (ESC)” refers to a stem cellderived from the inner cell mass of a blastocyst, an early-stage embryo.Human embryos reach the blastocyst stage 4-5 days post fertilization, atwhich time they consist of 50-150 cells.

The term “progenitor cell” as used herein refers to an undifferentiatedcell that has the ability to proliferate and differentiate into one ormore different cell lineages, but is thought to have no or limitedability to self renew. Typically, a stem cell culture, such as, e.g., anhESC culture or NSC culture, will contain some progenitor cells inaddition to stem cells. In some instances, progenitor cells derive fromstem cells, during the process losing the ability to self-renew butmaintaining the ability to differentiate into one or more different celllineages. In other words, stem cells can give rise to progenitor cells.

As used herein, the terms “neural stem cell” and “neural progenitor cell(NPC)” describes undifferentiated cells that can generate nervous systemcells. NSCs have the ability to self renew, whereas NPCs are thought tohave very limited or no ability to self renew.

As used herein, a “retinal pigment epithelial stem cell (“RPESC”) is astem cell that is activated from the adult human retinal pigmentepithelium (RPE).

As used herein, induced pluripotent stem cells (“iPSCs”) are pluripotentstem cells expressing many of the genetic and phenotypic characteristicsof ES cells, and are derived from differentiated (e.g., somatic). iPSCshave the same gross morphology as ES cells, proliferative properties,form teratomas after transplantation into nude mice, and have theability to differentiate along all 3 germ layers in vitro. Theirresponses to key factors such as retinoic acid and leukemia inhibitoryfactor (LIF) are also the same as those observed for ES cells [see,Okita et al, 2007; Nature 448: 313-317].

As used herein, “mesenchymal stem cells (MSCs)” are multipotent stemcells that can be derived from a variety of tissues and candifferentiate into a variety of cell types, including osteoblasts (bonecells), chondrocytes (cartilage cells) and adipocytes (fat cells) andare capable of self renewal [see, Pittinger et al. Science (1999) 284;143-7; Herzog (2003) Blood; 102; 3483-93]. MSCs have been characterizedby a number of surface markers including expression of markers from thefollowing list: CD29, CD44, CD73, CD105, CD106, CD166 and STRO-1 [see,Xu et al; Cell Research (2007) 17; 240-248; Simmons and Torok-Storb(1991) Blood; 78:55-62].

Hematopoietic stem cells (“HSCs”) are multipotent stem cells that giverise to all the blood cell types from the myeloid (monocytes andmacrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes/platelets, dendritic cells), and lymphoid lineages(T-cells, B-cells, NK-cells), and are capable of self renewal. Mouse andhuman HSCs have been identified by different combinations of markers.Mouse HSCs typically express c-Kit and Sca-1 but are negative formarkers of mature hematopoietic cell lineages (Lin−) [see, Challen etal. Cytometry A; 2009; 75; 14-24]. Human HSCs have been described asCD34+ CD133+ Lin− cells [see, Hawley et al, 2006 Methods in Enzymol419:149-179].

As used herein, a “cancer stem cell (CSC)” is a cancer cell (typicallyfound within tumors or hematological cancers) that possesses the abilityto give rise to all cell types found in a particular cancer sample andthe ability to self renew. These cells are also termed ‘tumor initiatingcells’, as these are recognized to have stem cell properties. Typicallycancer stem cells share markers of stem cells of the tissue of origin,thus leukemia stem cells can be identified as CD34+ CD38− and breastcancer stem cells can be identified as CD24-CD44+ [see, Bonnet and Dick;Nature Medicine (1997) 3:730-737; Reya et al. (2001) 414:105-111; Kai etal. (2010) Breast cancer; 17:80-85; Gibson et al.; Nature; 2010;468:1095-9].

The term “growth factor” can be a naturally occurring, endogenous orexogenous protein, or recombinant protein, capable of inhibiting and/orstimulating differentiation of cells, such as e.g., stem or progenitorcells. The term “growth factor” also can encompass lipid, chemical, andother non-protein agents, e.g., small molecules that are capable ofinhibiting and/or stimulating cell differentiation. In certainembodiments, the term “growth factor” refers to any polypeptide or otheragent that is capable of inhibiting or stimulating cell differentiation,e.g., when present in effective amounts in a stem or progenitor cellculture. Growth factor polypeptides of the present invention includeboth naturally occurring and recombinant proteins, which may be eitherendogenous or exogenous to the cells being cultured. In addition, agrowth factor of the invention may be a synthetic protein, such as afusion or other protein construct or a chemical modification of theamino acid sequences derived from a naturally occurring growth factor orother protein. Such growth factors may be used in combination, toproduce, e.g., an additive or synergistic effect, according to thepresent methods.

A preferred growth factor used in the present invention is known as“basic fibroblast growth factor (bFGF)” or, alternatively, as“fibroblast growth factor 2 (FGF2).” The terms bFGF and FGF2 aresynonymous and refer to full-length proteins or any functionally activefragment thereof, which can be an isolated, naturally occurring form ofFGF2 or a recombinant form. Active fragments, mutant forms and chemicalmodifications of FGF2 that retain the functional properties of wild-typeFGF2 (specifically, the ability to maintain cells in an undifferentiatedstate), are contemplated for use in the methods of the presentinvention. Modifications of growth factor for the stabilization of thegrowth factor are described in detail, for example, in Caccia et al.(1992) European Journal of Biochemistry, 204: 649-655. Caccia et al.describe stabilized forms of recombinant human basic fibroblast growthfactor by chemical modifications of cysteine residues. Formulations forstabilizing FGF2 are also described in U.S. Pat. No. 5,217,954 by Fosteret al.

As used herein, the terms “mutant” and “mutation” refer to anydetectable change in genetic material (e.g., DNA) or any process,mechanism, or result of such a change. This includes gene mutations, inwhich the structure (e.g., DNA sequence) of a gene is altered, any geneor DNA arising from any mutation process, and any expression product(e.g., protein or enzyme) expressed by a modified gene or DNA sequence.As used herein, the term “mutating” refers to a process of creating amutant or mutation. Exemplary sequences of FGF2 polypeptides that may beused in the present invention are known in the art and are available,e.g., from the GenBank® database. For example, the amino acid sequenceof an exemplary human FGF2 polypeptide is available under the GenBank®Accession No. NP_(—)001997 (SEQ ID NO: 1), whereas the amino acidsequence of an exemplary mouse FGF2 polypeptide is available fromGenBank® under the Accession No. AAP92385 (SEQ ID NO: 3). Sequences ofexemplary nucleic acids encoding these human and mouse FGF2 polypeptidesequences are also available from GenBank®, under the Accession Nos.NM_(—)002006 (SEQ ID NO: 2) and NM_(—)008006 (SEQ ID NO: 4),respectively.

FGF2 polypeptides that may be used in the present invention may also beobtained from commercial sources, such as from, e.g., R&D Systems(Minneapolis, Minn.); Peprotech, (Rocky Hill, N.J.); Becton Dickinson,(San Jose, Calif.); Invitrogen, (Carlsbad, Calif.). Recombinant FGF2 mayalso be expressed in cells using any suitable expression system known inthe art for producing recombinant protein. Recombinant and/or naturallyoccurring FGF2 can be isolated using any suitable technique known in theart.

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced.Isolated nucleic acid molecules include, for example, a PCR product, anisolated mRNA, a cDNA, or a restriction fragment. Isolated nucleic acidmolecules also include, for example, sequences inserted into plasmids,cosmids, artificial chromosomes, and the like. An isolated nucleic acidmolecule is preferably excised from the genome in which it may be found,and more preferably is no longer joined to non-regulatory sequences,non-coding sequences, or to other genes located upstream or downstreamof the nucleic acid molecule when found within the genome. An isolatedprotein can be associated with other proteins or nucleic acids, or both,with which it associates in the cell, or with cellular membranes if itis a membrane-associated protein.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for example,producing an non-coding (untranslated) RNA or a protein by activatingthe cellular functions involved in transcription and translation of acorresponding gene or DNA sequence. A DNA sequence is expressed in or bya cell to form an “expression product” such as RNA or a protein. Theexpression product itself, e.g. the resulting RNA or protein, may alsobe said to be “expressed” by the cell. The term “expression system”means a host cell and compatible vector under suitable conditions, e.g.for the expression of a protein coded for by foreign DNA (the“expression construct”) carried by the vector and introduced to the hostcell. By “expression construct” is meant a nucleic acid sequencecomprising a target nucleic acid sequence or sequences whose expressionis desired, operatively associated with expression control sequenceelements which provide for the proper transcription and translation ofthe target nucleic acid sequence(s) within the chosen host cells. Suchsequence elements may include a promoter and a polyadenylation signal.The “expression construct” may further comprise “vector sequences”. By“vector sequences” is meant any of several nucleic acid sequencesestablished in the art which have utility in the recombinant DNAtechnologies of the invention to facilitate the cloning and propagationof the expression constructs including (but not limited to) plasmids,cosmids, phage vectors, viral vectors, and yeast artificial chromosomes.By “operatively associated with” is meant that a target nucleic acidsequence and one or more expression control sequences (e.g., promoters)are physically linked so as to permit expression of the polypeptideencoded by the target nucleic acid sequence within a host cell.

Expression constructs of the present invention may comprise vectorsequences that facilitate the cloning and propagation of the expressionconstructs. A large number of vectors, including plasmid and fungalvectors, have been described for replication and/or expression in avariety of eukaryotic and prokaryotic host cells. Standard vectorsuseful in the current invention are well known in the art and include(but are not limited to) plasmids, cosmids, phage vectors, viralvectors, and yeast artificial chromosomes. The vector sequences maycontain a replication origin for propagation in E. coli; the SV40 originof replication; an ampicillin, neomycin, or puromycin resistance genefor selection in host cells; and/or genes (e.g., dihydrofolate reductasegene) that amplify the dominant selectable marker plus the gene ofinterest. For example, FGF2 can be expressed using E. coli bacteria anddoes not need to be modified posttranslationally to be active. Anygrowth factors encompassed by the present invention can be expressed asrecombinant protein, or isolated from a naturally occurring source, orpurchased commercially, when available.

As used herein, the term “sustained release of a growth factor” from asustained release composition, in the context of a cell culture, meansthe growth factor is released from the sustained release composition(i.e., is made available to the cells in the cell culture) over a periodof time, preferably over at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, or at least 7days, or longer, e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks or longer, andthe released growth factor is maintained at a constant concentration, orrelatively constant concentration (i.e., a “stable concentrationrange”), in the cell culture. The stable concentration range ispreferably within about 50% of the starting growth factor concentration.Hence, in the context a cell culture, the growth factor is preferablyreleased from the sustained release composition such that theconcentration or amount of growth factor available to the cells(preferably a period of at least 1-7 days, as set forth above) is within50% of the amount or concentration of the growth factor that isavailable to the cells at the beginning of that time period. In evenmore preferable embodiments, the stable concentration range is not lessthan about 60%, 70%, 80%, 90% or 95% of the starting growth factorconcentration. In particularly preferred embodiments, the concentrationof released growth factor stays within the range of about 80%-100%,80%-95%, or 80%-90% of the starting concentration of the growth factorover a period of 1 day or more. In other preferred embodiments, theconcentration of released growth factor stays within the range of atleast 80%-100%, 80%-95%, or 80%-90% of the starting concentration of thegrowth factor over a period of 3 days or more. Sustained release of agrowth factor, such as FGF2, can be confirmed by detecting the levels ofthe growth factor in the cell culture over time (e.g., by ELISA).Sustained release of a growth factor may also be referred to herein as“time release” of the growth factor, or stabilization of the growthfactor, and includes any means or method of maintaining a stableconcentration range of released growth factor (e.g., in a cell culture).

A “sustained release composition” of the invention can include anysuitable vehicle that results in the maintenance of a stableconcentration range of one or more released factors (e.g., growthfactors) over a period of time. As used herein, sustained releasecompositions are suitable for culturing with undifferentiated cells,such as but not limited to stem and/or progenitor cells. Non-limitingexamples of sustained release compositions of the invention includemicrospheres (e.g., poly(DL-lactide-co-glycolide) (PLGA) microspheres),anhydrous poly-vinyl alcohol (PVA), millicylinders, alginate gels,biodegradable hydrogels, complexing agents and nanoparticles. [See,e.g., Ashton, et al. (2007) Biomaterials, 28, 36, 5518; Drury, J. L. etal. (2003) Biomaterials; 24:4337-4351; U.S. Pat. No. 7,226,617 to Dinget al.; Simmons, C. A. et al. (2004) Bone; 35:562-569; Zhu, G. et al.(2000) Nat Biotech; 18:52-57, Biodegradable Hydrogels for Drug Delivery,K. Park et al, 1993, Technomic Publishing, Trans Am Ophthalmol Soc, K.Derwent et al, 2008; 106:206-13.] Mechanical means and methods fortime-release are also included, for example manual addition or amechanical device (e.g. robotic) can be used to provide a continuous, ornear continuous, sustained supply of growth factor to a cell cultureover time and thereby maintain the stem or progenitor cells at a stablelevel of differentiation due to exposure to a stable concentration ofgrowth factor. Modifications of the growth factor or other means toreduce degradation and thereby result in a sustained concentration rangeof growth factor exposed to the stem cell culture are also included.

As used herein, the term “maintains the cell in an undifferentiatedstate” refers to preventing or minimizing the amount of celldifferentiation, e.g., spontaneous differentiation in culture. Incertain embodiments, such as, e.g., when culturing stem or progenitorcells, the term also includes maintaining the immature state whichincreases the ability of the stem cell to differentiate into one or moremature, differentiated cell lineages. For example, a multipotent stemcell or progenitor cell that is maintained in an undifferentiated statewill express markers associated with stem or progenitor cells, but willexpress no or low levels of markers associated with differentiating ordifferentiated cells, and will also maintain its ability todifferentiate into one more different cell lineages, i.e., will remainmultipotent. Thus, a unipotent keratinocyte progenitor cell that ismaintained in the undifferentiated state, for example, will notdifferentiate into a keratinocyte (i.e., will remain a progenitor cell),but will maintain the ability to differentiate into a keratinocyte(e.g., under appropriate culture conditions that signal the cell toundergo such differentiation).

Generally, it is possible to determine if a stem or progenitor cell is“maintained” as a stem or progenitor cell (i.e., maintained in anundifferentiated state) by determining whether it continues to expressone or more markers associated with such cells. For example, markers ofhuman embryonic stem cells, include, but are not limited to the markersOCT4, NANOG, TRA-1-81, SOX2, SSEA-4, and/or SSEA-3. Markers of NSCs andNPCs include without limitation, Nestin, Lex (CD-15), Musashi, Bmi-1,Sox1, Hes1, Hes5, BLBP, and CD133. In addition, an ESC that ismaintained in an undifferentiated state generally will not express, orwill express relatively low levels of markers indicative ofdifferentiation such as Brachyury, Sox17, Foxa2, Pax6, Otx2, and Sox1.An NSC that is maintained in an undifferentiated state will not express,or will express relatively low levels (compared to a differentiatedcell) of markers including without limitation, Tuj1, S100β,galactocerebroside and/or MBP (myelin basic protein). For instance, asshown in the present Examples, the growth factor FGF2 maintains hESCs inan undifferentiated state, as evidenced by the high expression of SSEA-4and OCT4 and low expression of the differentiation markers Brachyury andSOX17. Markers such as SSEA-4, OCT4, Brachyury and Sox17, as well asother markers indicative of differentiated and undifferentiated states,are known and routinely used in the art. Maintaining theundifferentiated state is critical for both deriving and maintainingstem cells.

Other undifferentiated cells encompassed by the present invention willalso express similar and/or different markers characteristic of theundifferentiated state and/or of a differentiated state of the cell.Such markers are known or may be readily determined by the skilledartisan, and the expression of such markers may be analyzed to determinewhether the cell is maintained in an undifferentiated state.

As used herein, the term “in the presence of a sustained releasecomposition” with respect to a cultured cell, means that the cell isable to be contacted by sustained concentrations of the growth factor orother agent provided by the sustained release composition or othermethod of maintaining stable growth factor concentration over time. Thecell and the sustained release composition may or may not be containedin the same well or other culture container. A cell is in the presenceof a sustained release composition including, e.g., if the sustainedrelease composition and the cell are separated from each other by atranswell or other divider, so long as fluid (e.g., culture media)and/or growth factors can be exchanged readily between the separatedareas containing the cell and the sustained release composition.

The term “about” or “approximately” means within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, still morepreferably within 10%, and even more preferably within 5% of a givenvalue or range. The allowable variation encompassed by the term “about”or “approximately” depends on the particular system under study, and canbe readily appreciated by one of ordinary skill in the art.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989 (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); Ausubel, F. M. et al.(eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc.,1994. These techniques include site directed mutagenesis as described inKunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985), U.S. Pat. No.5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360(1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh andGuengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech.13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang andMalcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641(1999), U.S. Pat. Nos. 5,789,166 and 5,932, 419, Hogrefe, Strategies 14.3: 74-75 (2001), U.S. Pat. Nos. 5,702,931, 5,780,270, and 6,242,222,Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson,Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996),Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch andJoly, Nuc. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J.Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993),Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al.,Meth. Molec. Biol. 67: 209-218. The skilled person will know and be ableto use these and other techniques routine in the art to practice thepresent invention.

II. Overview

As discussed supra, present methods for culturing undifferentiatedcells, such as stem and/or progenitor cells, are faced with at least twosubstantial difficulties. First, growth factors, such as FGF2, must beadded frequently to the cultures, thereby making the culture of suchcells a time consuming, laborious and expensive undertaking Standardprotocols that add growth factor daily for ESC and every three days forNSC result in significant variations of growth factor concentrations towhich the cells are exposed. Second, in the case of culturingundifferentiated cells such as stem cells and/or progenitor cells, asignificant amount of unwanted, spontaneous differentiation of the cellsoccurs during culturing, even when growth factors such as FGF2 areadministered daily to inhibit such differentiation. Hence, not only doesthe invention substantially reduce the labor and effort required toculture and maintain undifferentiated cells in an undifferentiatedstate, the resulting cultures exhibit surprisingly less spontaneousdifferentiation (resulting in less heterogeneity in the culture), andare therefore of unexpectedly improved quality, and therefore, utility,compared to cells cultured by currently available methods.

As an instance, and not by way of limitation, the Examples providedherein describe experiments where hESCs, iPSCS, RPESCs and NSCs werecultured using a sustained release composition containing a stable formof the growth factor FGF2 that provided a stable concentration range ofFGF2 protein for up to 7 days or more (e.g., up to 35 days), byminimizing variation of growth factor concentration (e.g., as shown inExample 2, FGF2 concentration in the cell cultures was maintained in therange of 80-100% over 3 days in cultures containing FGF2-containingmicrospheres), significantly improving the stability of undifferentiatedhESCs, iPSCS, RPESCs, NSCs and NPCs. In one Example, hESCs cultured inthe presence of a sustained release composition containing FGF2 weremaintained in an undifferentiated state in the absence of MEFs or MEFconditioned media. Furthermore, sustained release of FGF2 greatlyimproved the pluripotency of a cell culture that had undergonedifferentiation (as shown in Example 6).

Thus, in certain aspects, the present invention provides novel methodsfor maintaining sustained, stable concentration ranges of one or moregrowth factors in biologically active form over an extended period oftime, preferably over a period of one or several days, and morepreferably for at least a week to maintain the cells in a moreundifferentiated state than standard methods of cell culture canachieve.

It was also demonstrated in the present Examples that frequent manualadministration of growth factor directly to NSC cultures (every 8hours), rather than the standard protocol (every third day), alsoimproved the stability of the undifferentiated NSC culture (i.e., morecells were maintained in the undifferentiated state, as measured by theexpression of Nestin and Tuj1). Further, the absolute number of cellsproduced was significantly increased. Thus, in certain embodiments, thepresent invention provides improved methods for culturing cells, such asNSCs, by exposing the cells to a steady level of growth factors (i.e.,stable concentration range) to maintain the cells in an undifferentiatedstate and produced increased numbers of cells.

Although the invention is not limited by any particular theory ormechanism of action, it is believed that the present culture methodsensure a stable concentration range of growth factor over time (e.g., atleast one day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, at least 7 days, or longer) that moreclosely mimics the in vivo environment. Hence, in preferred embodiments,one or more growth factors may be delivered in sustained releaseformulations to a cell culture at the beginning of the culturingprocess, and no further media changes are required during the extendedtime period (e.g., for multiple days, and preferably for at least sevendays). In another embodiment, the growth factor, such as FGF2, may beadministered frequently to the cell culture, e.g., three or more timesper day or continuously, every day of the culture. The end result is amore homogeneous, undifferentiated cell culture (i.e., a more stableundifferentiated cell population) containing significantly more cells.

In a preferred embodiment, the undifferentiated cell is a stem cell orprogenitor cell; however, a person of skill in the art will appreciatethat the present methods are useful for culturing any undifferentiatedcell that requires frequent administration of a growth factor or otheragent.

III. Stem And Progenitor Cells

In certain embodiments, the present methods are useful for maintainingESCs, in an undifferentiated state in standard culture dish surfaces. Inanother aspect of the invention, the present methods are useful forrescuing an ESC culture from differentiation restoring differentiatedESCs back to an undifferentiated state by culturing the ESCs in thepresence of a sustained stable concentration range of growth factor,preferably FGF2. The present methods are useful for culturing anymammalian ESCs, such as but not limited to human, rat, pig, sheep,mouse, or non-human primate ESCs. ESCs can be derived according to anysuitable method known in the art [Thomson et al., 1998; Amit, 2000 andCowan et al., 2004; see also, U.S. Pat. Nos. 5,843,780; 6,200,806; and7,029,913 (all by Thomson)], or by modifications of those protocols toinclude sustained levels of growth factor(s), as described herein.

Human ESCs (hESCs) are characterized for example by high expression ofOctamer-binding protein 4 (Oct-4) (GenBank® Accession No.NM_(—)001159542.1 (mRNA), Swiss-Prot Reference No. Q01860.1 (protein));Nanog (GenBank® Accession Nos. NM_(—)024865.2 (mRNA), AAP49529.1(protein)); SRY (sex determining region Y)-box 2 (Sox2) (GenBank®Accession Nos. NM_(—)003106.3 (mRNA), NP_(—)003097.1 (protein)),TRA-18-1, SSEA-4, and SSEA-3 (see, Henderson, J K et al. (2002); StemCells; 20 (4):329-37 for description of expression of TRA-1-81, SSEA-4and SSEA-3 markers in human ESCs), and low expression of markers of hESCdifferentiation, such as Brachyury (GenBank® Accession Nos.NM_(—)080646.1 (mRNA, variant A), NM_(—)080647.1 (mRNA, variant C),NM_(—)005992.1 (mRNA, variant B), AAB94018.1 (protein)), SRY (sexdetermining region Y)-box 17 (Sox17) (GenBank® Accession Nos.NM_(—)022454.3 (mRNA), NP_(—)071899.1 (protein)), Forkhead box A2(Foxa2) (GenBank® Accession Nos. NM_(—)021784.4 (mRNA, variant 1),NM_(—)153675.2 (mRNA, variant 2), and AAH06545.2 (protein)); paired box6 (Pax6) (GenBank® Accession Nos. NM_(—)000280.3 (mRNA, variant 1),NM_(—)001604.4 (mRNA, variant 2), NM_(—)001127612.1 (mRNA, variant 3),and ABB55263.1 (protein)); orthodenticle homeobox 2 (Otx2) (GenBank®Accession Nos. NM_(—)021728.2 (mRNA, variant 1), NM_(—)172337.1 (mRNA,variant 2); NP_(—)068374.1 (protein, isoform a), NP_(—)758840.1(protein, isoform b); and SRY (sex determining region Y)-box 1 (Sox1)(GenBank® Accession Nos. NM_(—)005986.2 (mRNA), NP_(—)005977.2(protein)). Appropriate markers of ESCs of various species of origin areknown, and can be readily determined by one skilled in the art.

In one aspect of the invention, the present methods are also useful formaintaining neural stem and/or progenitor cells in an undifferentiatedstate in culture. In another aspect of the invention, the presentmethods are useful for rescuing a neural stem and/or progenitor cellculture from differentiation by culturing the cells in the presence ofsustained concentration range of growth factor, preferably FGF2.

NSCs and NPCs which may be cultured according to the methods describedherein can be identified by the expression of certain markers, such asone or more of Nestin (GenBank® Accession Nos. NM_(—)006617.1 (mRNA),NP_(—)006608.1 (protein)); fucosyltransferase 4 (alpha (1,3)fucosyltransferase, myeloid-specific) (FUT4) (LeX) (CD-15) (GenBankAccession Nos. NM_(—)002033.3 (mRNA), NP_(—)002024.1 (protein)); Musashi(GenBank Accession Nos. AB012851.1 (mRNA), BAA33962.1 (protein));polycomb complex protein Bmi-1 (Bmi-1) (GenBank Accession Nos.NM_(—)005180.8 (mRNA), NP_(—)005171.4 (protein)); Sox1 (GenBank®Accession Nos. NM_(—)005986.2 (mRNA), NP_(—)005977.2 (protein)); SRY(sex determining region Y)-box 2 (Sox2) (GenBank® Accession Nos.NM_(—)003106.3 (mRNA), NP_(—)003097.1 (protein)); Hest (GenBank®Accession Nos. Y07572.1 (mRNA), CAA68857.1 (protein)); HesS (GenBank®Accession Nos. DQ272660.1 (mRNA), ABB83829.1 (protein)); fattyacid-binding protein, brain (BLBP) (GenBank® Accession Nos.NM_(—)001446.3 (mRNA), NP_(—)001437.1 (protein)); and CD133 (GenBank®Accession Nos. NM_(—)006017.2 (mRNA, variant 1), NM_(—)001145847.1(mRNA, variant 2), NM_(—)001145848.1 (mRNA, variant 3),NM_(—)001145852.1 (mRNA, variant 4), NM_(—)001145851.1 (mRNA, variant5), NM_(—)001145850.1 (mRNA, variant 6), NM_(—)001145849.1 (mRNA,variant 7), NP_(—)006008.1 (protein, isoform 1), NP_(—)001139320.1(protein, isoform 2), NP_(—)001139324.1 (protein, isoform 4),NP_(—)001139323.1 (protein, isoform 5), NP_(—)001139322.1 (protein,isoform 6), and NP_(—)001139321.1 (protein, isoform 7), and no orrelatively low levels (compared to a differentiated cell) of markersincluding without limitation, Tuj1, S100β (GenBank® Accession Nos.,e.g., NM_(—)006272.2 (mRNA), NP_(—)006263 (protein) (other isoforms arealso described and available from GenBank®)), galactocerebroside(GenBank® Accession Nos. NM_(—)000153.3 (mRNA), NP_(—)000144.2(protein)) and/or MBP (myelin basic protein) (GenBank® Accession Nos.,e.g., NM_(—)001025081.1 (mRNA), and NP_(—)001020252 (protein) (otherisoforms are also described and available from GenBank®)).

As used herein, “neural” means the nervous system and includes glialcells and neurons. NPCs can also express high levels of helix-loop-helixtranscription factors NeuroD, Atoh1, and neurogeninl and neurogenin2 NSCcultures typically contain a mixture of NSCs and NPCs, and both may becultured according to the methods of the present invention. [See,Abramova et al, Developmental Biology 2005 283:269-81;Beckervordersandforth, Cell Stem Cell (2010) 7:744-758; Shoemaker etal.; Plos One (2010) 5:e9121.]

NSCs and NPCs have the potential to differentiate into neural cells,such as, e.g., neurons, glia, astrocytes, retinal neurons,photoreceptors, oligodendrocytes, olfactory cells, hair cells,supporting cells, and the like.

In certain aspects, the present invention provides methods that areuseful for culturing undifferentiated cells, such as ESCs, NSCs andNPCs, as well as related cell types. For example, the blastocystcontains parts that yield stem cells known as epiblast stem cells, andthe nervous system contains several subtypes of NSCs. The advantage ofthe sustained release methods over daily or every 3 day administrationmethods of the present invention for these subtypes is expected to bethe same as for ESCs, NSCs and NPCs. In other aspects, the presentinvention provides methods for culturing other undifferentiated cells,such as e.g., immature neural cells, wherein the cell is maintained inthe undifferentiated state. In some aspects, in addition to ESCs, NSCsand NPCs, as well as iPSCs, MSCs and CSCs undifferentiated cells caninclude, without limitation, stem and/or progenitor cells of the skin,hair, gut, and blood, as well as other stem and/or progenitor cellsincluding adipose, renal, epiblast, and bone marrow stem and/orprogenitor cells, among many others.

In other aspects, the present invention provides methods that are usefulfor culturing undifferentiated cells, such as iPSCs, RPESCs, HSCs, MSCs,and CSCs. RPESCs can be expanded many fold in vitro and produce a widevariety of progeny from diverse developmental lineages (includingmesoderm and ectoderm). RPESCs are capable of producing retinal cells,and they also are capable of producing a much wider repertoire ofprogeny, including bone, muscle and adipocytes. These cells and how toidentify and/or isolate them are described in detail in U.S. PatentApplication Publication No. 2009/0274667 by Temple et al. Such cells canalso be cultured according to the methods of the present invention.

iPSCs are useful for both in vitro study of stem cells (e.g., factorscontrolling stem cell differentiation) and for the application of iPSCsfor the treatment of disease. iPSCs can be derived from murine and humanfibroblasts by introducing four specific transcription factors, SOX2(GenBank® Accession Nos. NM_(—)003106.3 (mRNA), NP_(—)003097.1(protein)), OCT4 (GenBank® Accession No. NM_(—)001159542.1 (mRNA),Swiss-Prot Reference No. Q01860.1 (protein)), Kruppel-like factor 4(gut) (KLF4) (GenBank® Accession Nos. NM_(—)004235.4 (mRNA) andNP_(—)004226.3 (protein)), and myc proto-oncogene protein (“c-MYC”)(GenBank® Accession Nos. NM_(—)002467.4 (mRNA) and NP_(—)002458.2(protein)), into the fibroblasts by viral transduction. [See, Lowry, W.E., et al. (2008). Proc. Natl. Acad. Sci. USA 105, 2883-2888; Maherali,N., et al. (2007). Cell Stem Cell; 1, 55-70; Park, et al. (2008). Nature451, 141-146; Takahashi, K., and Yamanaka, S. (2006). Cell; 126,663-676; Takahashi, K. et al. (2007). Cell 131, 861-872; and Yu, J. etal. (2007). Science; 318, 1917-1920; Stadtfeld and Hochedlinger (2010)Genes Dev; 24:2239-2263; Hochedliner and Plath (2009) 136; 509-523.] Ithas also been reported that OCT4, SOX2, NANOG, and LIN-28 (GenBank®Accession Nos. NM_(—)024674.4 (mRNA), and NP_(—)078950.1 (protein)) aresufficient to reprogram human somatic cells into pluripotent stem cells.Importantly, FGF2 is also used in the process of reprogrammingfibroblast to the pluripotent state (i.e., for deriving iPSCs fromfibroblasts). Furthermore, in culture, iPSCs are dependent on FGF2 inorder to be maintained in an undifferentiated state. [See, Takahashi etal. Cell, Volume 131, Issue 5, 861-872, 30 November 2007.]

MSCs can be obtained from a variety of tissues from the embryo throughadulthood. For example, they can be extracted from the umbilical cordtissue, namely Wharton's jelly and umbilical cord blood or from amnioticfluid. MSCs can also be obtained from the developing tooth bud of themandibular third molar, from the bone marrow, skeletal muscle,poeriosteium, lung, dermis and from adipose tissue. MSCs are widelypresent in the adult and can be extracted from a great variety oftissues. [See, Lu et al, Haematologica, 2006 91: 1017-26; Zeddou et al,Cell Biol Int 2010. vol 34:693-701; Lee et al Cell Physiol Biochem 200414:311-324; Rosenbaum et al 2008; Organogenesis 4:23027.] Rosenbaum etal. describes surface markers that can be used to identify MSCs.

Cancer stem cells (CSCs) are tumorigenic and may generate tumors throughthe stem cell processes of self-renewal and differentiation intomultiple cell types. CSCs are thought to persist in tumors as a distinctpopulation that can cause relapse and metastasis by giving rise to newtumors. Development of specific therapies targeted at CSCs are needed,e.g., for the treatment of cancer. Thus, it is useful to culture thesecells in order to study them and to develop such therapies. However,CSCs are often very difficult to culture from primary tumor samples,illustrating their dependence on the growth factor environment found invivo. Thus, improved methods for their culture are needed. CSCs can becultured according to the present invention in the presence of asustained concentration range of growth factor, in order to maintainthem in an undifferentiated state. Primary tumors from a variety ofsources are frequently FGF2 dependent and, in some cases, show superiorgrowth in FGF2 [see, Lee et al, Cancer Cell (2006), vol 9 391-403;Ricci-Vitiani et al; Nature; 445 pp 111-115; New Yeoman, J Cell OPhysiol(1992) 150:320-326].

Hematopoietic stem cells (HSCs) can be isolated from bone marrow orperipheral blood. Methods for isolation of HSCs are described, e.g., inHawley et al; Methods Enzymol; 2006; 419:149-179; and Challen et al,Cytometry A; 2009; 75; 14-24. HSCs are very difficult to culture exvivo. However, FGF2 can help preserve their self-renewal [see, Yeoh etal, Stem Cells; 2006, 24:1564-1572.]

Non-limiting examples of stem cells and related progenitor cells thatmay be cultured according to the methods of the invention include, e.g.,skin stem cells, spermatagonial stem cells, hair follicle stem cells,cancer stem cells, bone marrow stem cells, gut stem cells, hematopoieticstem cells, adipose stem cells, mouse embryonic stem cells, humanembryonic stem cells, retinal pigment epithelial stem cells, mesenchymalstem cells, epiblast stem cells, renal stem cells, amniotic stem cells,umbilical blood stem cells, endothelial stem cells, and neural creststem cells. Progenitor cells related to the above stem cells (i.e.derived from such stem cells) may also be cultured according to thepresent invention. All stem cells requiring growth factor to maintainthe undifferentiated state. Any stem or progenitor stem cells now knownor to be discovered may also be cultured according to the methods of thepresent invention. Stem and progenitor cells may be derived by theskilled artisan, and such methods are known in the art. Many stem cellsare also commercially available or available from cell banks, such as,e.g., the WiCell Reseach Institute, National Stem Cell Bank, Madison,Wis.

IV. Cell Culture Methods

The present invention provides novel and improved methods for culturingundifferentiated cells, such as but not limited to stem and/orprogenitor cells. For culturing stem and/or progenitor cells,appropriate culture medium is known and described in the art. [See,e.g., Amit et al., 2000, supra; Fasano et al., 2010, supra; Ludwig etal., 2006, supra; Bendall, et al., 2007, supra; Qian et al., 1997,supra, Fasano et al., 2007, supra; and Shen Q, et al. (2004) Science304, 1338-40.] For example, cells can be cultured in serum freeDMEM/high-glucose supplemented with N2 and B27 solutions and growthfactors. Typically cells are incubated at 37° C., and 5% CO₂ in tissueculture treated wells. Optionally, cells can be cultured in the presenceof feeder cells, such as mouse embryonic fibroblasts (MEFs); however, asdemonstrated by the present Examples, the present invention providessustained release compositions that eliminate the need for feeder cells.Such methods are well known in the art. [See, e.g., Amit et al., 2000;Fasano et al, 2010; Ludwig et al., 2006; Bendall, et al., 2007; Qian etal., 1997, Fasano et al., 2007; and Shen et al., 2004.] Specific cultureconditions are readily determined and adjusted by the ordinarily skilledartisan.

Typically, ESCs are grown until density is deemed suitable for theappropriate experiments being carried out by the investigator (typicallyone week). At this point, ESCs are passaged either as single cells orcell aggregates onto MEF feeders, or tissue culture treated plasticdishes coated with an extracellular matrix (Fasano et al., 2010).Typically, NSCs and/or NPCs are grown until density is deemed suitablefor the appropriate experiments being carried out by the investigator(typically one week). At this point, NSCs and/or NPCs are passaged aseither single, dissociated cells or cell aggregates onto tissue culturetreated plastic dishes coated with an extracellular matrix or non-tissueculture treated plates with no extracellular matrix when floating NSC(neurosphere) cultures are needed (Fasano et al., 2007).

Standard culture methods for the culture of stem cells such as, but notlimited to, iPSCs, MSCs, CSCs and SCSCs are known in the art. Forexample, methods for the culture of iPSCs are described in detail, e.g.,in Maherali and Hochedlinger, Cell Stem Cell 2008 3:595-605. Methods forthe culture of MSCs are described in Sotiropoulou P A, et al.; StemCells. 2006 February; 24 (2):462-71. Methods for the culture of CSCs aredescribed, e.g., in Cao, L. et al. BMC Gastroenterol. 2011 Jun. 14; 11(1):71; and Wakimoto et al. Cancer Res. 2009 Apr. 15; 69 (8):3472-81.Methods for the culture of SCSCs are described, e.g., in Lowry et al.Exp Neurol. 2008 February; 209 (2):510-22; Yan J. et al., PLoS Med. 2007February; 4 (2):e39; and Yeoh et al, stem cells 2006, 24:1564-1572.

In the present methods, the undifferentiated cells are incubated in thepresence of sustained concentration range of growth factor. In certainembodiments, the undifferentiated cells are incubated in the presence ofa sustained release composition, such as, e.g., PLGA microspheres. Insome aspects, the undifferentiated cells can be incubated with two ormore sustained release compositions that are the same or differentcompositions containing the same or different growth factor(s). Incertain aspects, the cells are cultured in the constant presence of oneor more growth factors by frequent feeding with the growth factor (e.g.,continuously, or every 2, 4, 6, or 8 hours). Preferably cells are fedmore than every 8 hours with the desired growth factors. According tothe present methods, the stable concentration range of growth factorsare useful for maintaining cells, such as stem or progenitor cells, inan undifferentiated state and are also effective for increasing thenumber, homogeneity and quality of progeny produced.

For “frequent feeding” of growth factors to a cell culture (e.g., growthfactor added to culture every few hours or continuously), the growthfactor may be administered to the cell culture by any suitable method,such as e.g., by pipette or dropper, or may be dripped in by anautomatic cell feeding system (i.e., mechanically).

V. Sustained Release Compositions

The invention provides sustained release compositions that maintainsustained concentrations, or concentration range, of growth factors inthe cell culture media over an extended period of time (e.g., over aperiod of several days and, preferably, for at least seven days). It isto be understood that the present invention is not to be limited to anyone particular or even to several sustained release compositions, suchas those described herein. The methods of the present invention may bepracticed using any suitable sustained release composition orpreparation (e.g., tissue culture plate coating), so long as the growthfactor(s) provided by the sustained release composition or preparationis provided in a manner that results in sustained, stable concentrationranges of growth factor, as described herein.

In other embodiments, growth factors may be added directly to a cellculture, e.g., multiple times per day, or continuously. In a specificembodiment, FGF2 is administered three times per day, every 8 hours,directly to an NSC culture causing unexpected and significantimprovement in the differentiation state and number of cells compared tostandard methods of culture (feeding FGF2 to the cells every 3 days).

Non-limiting examples of sustained release compositions that may be usedin the methods of the invention include, e.g., microspheres (e.g.,poly(DL-lactide-co-glycolide) (PLGA) microspheres), anhydrous poly-vinylalcohol (PVA), millicylinders, alginate gels, biodegradable hydrogels,liposomes, complexing agents, continuous micropumps, nanoparticles andany biocompatible material that releases growth factor over sustainedperiods. [See, e.g., Ashton, et al. (2007) Biomaterials, 28, 36, 5518;Drury, J. L. et al. (2003) Biomaterials; 24:4337-4351; U.S. Pat. No.7,226,617 to Ding et al.; Simmons, C. A. et al. (2004) Bone; 35:562-569;Zhu, G. et al. (2000) Nat Biotech; 18:52-57, Biodegradable Hydrogels forDrug Delivery, K. Park et al, 1993, Technomic Publishing, Trans AmOphthalmol Soc, K. Derwent et al, 2008; 106:206-13; see also publishedU.S. Patent Application Publication No. 2010/0021422.] In the presentExamples, microspheres were prepared according to methods described inZhu, G. et al. (2000) Nat Biotech; 18:52-57, modified using a standarddouble emulsion technique.

In some aspects, microspheres or microparticles of the present inventionmay comprise a combination of PLGA and PVA. [See, Edlund et al.,Advances in Polymer Science Vol. 157, 2002, 67) lists on page 77 anumber of different degradable polymers investigated for controlled drugdelivery applications (e.g., polyglycolide, polylactide, etc.).] Thus,suitable sustained release compositions useful in the present inventioncould be made using these or other degradable polymers. PVA is one of arange of possible substances that can be used to stabilize microspheresproduced by emulsion solvent evaporation techniques. PVA is used as astabilizing/emulsifying agent. Varying the concentration of PVA canenable the size of the microspheres to be varied, which in turn caninfluence the release profile [see, e.g., Zhao et al. (2007) BioMagneticResearch and Technology; 5:2].

PLGA microspheres of the present invention can range in size from 10-40μIna, with an average diameter of 20 μm. The size can be controlled byvarying the speed of the homogenizer, etc. In certain embodiments,larger particles can be used; varying the size of the microspheres canbe guided by culture condition considerations, the desired kinetics ofrelease, loading efficiency and amount of growth factor release.

In some embodiments, PLGA millicylinders may be used as a sustainedrelease composition of the invention for the time release of growthfactor(s) to cells in culture. As used herein, “millicylinders” aresingle cylindrical implants approximately 0.8-1.5 mm in diameter. [See,Zhu, G. et al. (2000) Nat Biotech; 18:52-57.] Yet another aspect of theinvention includes the use of nanoparticles for the time release ofgrowth factor(s) to cells, such as undifferentiated cells, in culture.As used herein, “nanoparticles” are defined as small particles that aretypically sized in the range of 1-100 nanometers (nm), but also includesub-micron as well as larger particles encompassing the range of 1-1000nm. [See, for example, Ya-Ping Li, et al., J. of Controlled Release, 71,2001, pages 203-211; Mu, L. and Feng, S.; J. of Controlled Release; 86,2003, 33-48; and Govender et al., J. of Controlled Release; 57, 1999,pages 171-185.]

Another aspect of the invention includes the suspension of microspheresin a hydrogel, which is considered biocompatible, biodegradable, and iscompatible with cells in cell culture, such as stem and/or progenitorcells. The hydrogel matrix has multiple uses including but not limitedto stabilizing in vivo applications of undifferentiated cells (e.g.,stem and/or progenitor cells), generating three-dimensional cellcultures, and delivering drugs, growth factors and other agents intocell culture. The bioactive factor is released from the microspherepresent in the hydrogel. Therefore, its rate of release can be adjustedin the same way as when there is no hydrogel (e.g., by changing thecomposition/molecular weight of polymers used to make the microsphere,changing protein loading in the microsphere, microsphere size, etc.). Itwas shown in Ashton et al. (supra), that the incorporation ofmicrospheres containing alginate lyase into the hydrogel enablecontrolled release of this enzyme which in turn provides control overthe rate of degradation of the hydrogel. [See, Ashton et al. (2007)(supra); Piantino et al., 2006, Exp Neurol., 201 (2):359-67; see also,U.S. Pat. No. 7,226,617 to Ding et al.]

Another example of a sustained release composition of the inventionincludes an amorphous carbohydrate glass matrix, as described in detailin PCT publication number WO 93/10758, in which a bioactive agent suchas FGF2 is incorporated into the carbohydrate glass matrix andcontrolled release or degradation is adjusted by addition of ahydrophobic substance. The present invention also provides coatingplastic or glass culture dishes with a matrix that releases growthfactor in a sustained manner. For example, the tissue culture plate timereleases FGF2 and/or other growth factors coated or contained in thesurface of the tissue culture plate or other container, over at leastabout 3, 4, 5, 6, or 7 days.

Typical concentrations of a sustained release composition (e.g., PLGAmicrospheres) for cell culture range from about 1 to about 300 ng/ml,preferably from about 1 to about 200 ng/ml, more preferably from about 1to about 100 ng/ml, even more preferably from about 1 to about 50 ng/ml,and most preferably from about 1 to about 10 ng/ml or from about 1 toabout 5 ng/ml. Typically, although not necessarily, about 7 μl per 1 mlof culture media of PLGA microsphere preparation (concentration 1000microspheres per μl of preparation) are added to the culture.

The present invention contemplates the use of any growth factor in thesustained release compositions of the invention that is useful formaintaining cells in an undifferentiated state. Further, a sustainedrelease composition of the invention may comprise two or more growthfactors.

A preferred growth factor that may be used in the present invention isFGF2. However, other non-limiting examples of suitable growth factors orother cytokines include, e.g., epidermal growth factor (EGF),platelet-derived growth factor (PDGF), sonic hedgehog (Shh), leukemiainhibitory factor (LIF) and Wnt proteins (e.g., Wnt1 or Wnt3) or anygrowth factor that maintains the stem cell in an undifferentiated state.For example, spinal cord stem cells (SCSCs) are expanded by Shh [see,Lowry, N. et al. Exp Neurol. 2008 February; 209 (2):510-22]. Thus, inone embodiment, SCSCs are cultured in the presence of a stableconcentration range of Shh in order to maintain the SCSCs in anundifferentiated state. In one embodiment, the SCSCs are cultured in thepresence of a sustained release composition, e.g., PLGA microspheres orother composition, such as but not limited to those described herein,wherein the sustained release composition comprises Shh. Additionally,the sustained release composition can comprise one or more of Mg(OH)₂,heparin, dextran sulfate, and EDTA.

Concentrations of growth factors in the sustained release compositionsof the invention can range from about 5% (w/v) to about 0.001%, fromabout 3% to about 0.05%, from about 2% to about 0.01%, and from about1.0% to about 0.1%. In a specific embodiment, a sustained releasecomposition comprises about 0.5% (w/v) FGF2. However, more or less FGF2can be used depending on the specific culture conditions.

Sustained release compositions of the invention may further compriseagents that stabilize and or complex with one or more growth factorscontained in the composition. For example, the composition may furthercomprise heparin, which has been shown to stabilize FGF2. [See,Caldwell, M. et al. (2004); Exp Neurol; 188:405-420.] Another agent thatcan be included in the composition in place of or in addition to heparinis dextran sulfate. Concentrations of heparin and/or dextran sulfate canrange from about 5% (w/v) to about 0.001%, from about 3% to about 0.05%,from about 2% to about 0.01%, and from about 1.0% to about 0.5%. In aspecific embodiment, a sustained release composition comprises about1.0% (w/v) heparin and/or dextran sulfate. However, more or less heparinor dextran sulfate can be used depending on the specific cultureconditions. In addition, other poly-anionic molecules that complex withFGF2 may be part of the present invention [J. Med. Chem., 2000, 43 (13),pp 2591-2600]. Covalent modifications of the growth factor that resultin reduced degradation are also included. All modifications that resultin sustained concentrations of growth factor in the stem cell growthmedia are expected to produce the same unexpected and surprising resultof improved proliferation, more homogenous undifferentiated populationof stem cells, and less stem cell differentiation.

Sustained release compositions of the invention may further compriseagents that modify the pH of the sustained release composition. Forexample, in a specific embodiment, Mg(OH)₂ is added to the compositionto neutralize an acidic environment, e.g., of a PLGA microsphere.Concentrations of Mg(OH)₂ can range from about 15% (w/v) to about 0.05%,from about 10% to about 0.1%, and from about 5% to about 1.0%. In aspecific embodiment, a sustained release composition comprises about3.0% (w/v) Mg(OH)₂. However, more or less Mg(OH)₂ can be used dependingon the specific culture conditions. The compositions of the inventionmay also optionally comprise agents such as e.g., EDTA, gum arabic,sucrose, MgCO₃, and BSA.

In a specific embodiment of the invention, a sustained releasecomposition is a PLGA microsphere, having a diameter of about 10-50microns, comprising 0.5% FGF2, 3% Mg(OH)₂, 1.0% heparin, and 1 mM EDTA.In another embodiment, a sustained release composition is a PLGAmicrosphere, having a diameter of about 10-50 microns, comprising 0.5%FGF2, 3% Mg(OH)₂, 1.0% dextran sulfate, and 1 mM EDTA. In eitherembodiment, the sustained release composition releases FGF2 over aperiod of at least about 1, 2, 3, 4, 5, 6, or 7 days, or longer, e.g., 2weeks, 3 weeks, 4 weeks, 5 weeks, or longer. Preferably, the compositionreleases FGF2 over at least 3 days or longer.

The kinetics and quantities of release of a growth factor from asustained release composition will depend on the specific compositionand size of the sustained release composition, the loading efficiency,the unreleased reservoir, the degradation rate of the growth factor aswell as the concentration of the growth factor and other agentscontained in the composition. The release kinetics and amount of growthfactor released, e.g., into the cell culture, are readily determined bythe skilled artisan. Preferably, a sustained release compositionreleasing FGF2 will release about 10 ng/ml FGF2 per day over at leastabout 3 to 7, 4 to 7, or 5 to 7 days for native FGF2 although less isneeded for stabilized forms of FGF2. For example, given 1 mg of FGF2microspheres in 1 ml of PBS containing 0.2% Tween 20, 1% BSA, 10 ug/mlheparin, and 1 mM EDTA, and based on 0.2% release of protein, 10 ng/mlof FGF2 will be achieved in the culture medium per day over a period ofat least about 3 days. The level of FGF2 and other growth factors neededto stabilize undifferentiated cells (i.e., maintain them in anundifferentiated state) may depend on the release kinetics anddegradation kinetics; however, preferably, the concentration of FGF2 inthe culture is maintained in the range of from about 1 ng/ml to about 10ng/ml or higher, e.g., 15 ng/ml or 20 ng/ml, or higher, over about 1day, 2 days, 3 days, 4 days, 5 days, 6 days 7 days, or longer, e.g., 2weeks, 3 weeks, 4 weeks, 5 weeks, or longer.

V. Assaying Cell Differentiation

A variety of methods can be utilized to determine whether anundifferentiated cell has been maintained as an undifferentiated cell.For example, cells in hESC cultures can be examined for the expressionof markers of undifferentiated cells, such as, but not limited to, OCT4,NANOG TRA-1-81, SSEA-4, and/or SSEA3. NSCs and NPCs can be examined forexpression of one or more markers such as, but not limited to, Nestin,Lex (CD-15), Sox-1, Sox-2, CD133, Musashi, Bmi-1, Hes1 and/or Hes5.

Such cells may also be examined for expression of markers ofdifferentiating or differentiated cells, such as, but not limited to,Brachyury, Sox17, Foxa2, Otx2, Sox1 and/or SSEA-1 (for hESCs); and suchas Tuj1, S100β, O4, and myelin basic protein (MBP) (for NSCs and NPCs).Gene and/or protein expression of such markers can be determined, e.g.,by quantitative PCR, FACS analysis, and/or ELISA. Such methods are wellknown in the art. Such markers are also useful for characterizingdifferentiating cells of other non-human species of origin, andappropriate markers are known and readily determined by the ordinaryskilled artisan.

It is to be understood that the methods of the present invention can beused for culturing many types of undifferentiated cells, such as but notlimited to stem cells, progenitor cells, neural cells such as immatureneural cells, etc., from any species of origin. A person of skill in theart can readily determine which markers are appropriate forcharacterizing the specific cell being cultured.

For stem cells, specifically, assessment of stem cell differentiation(or maintenance of stem cells in an undifferentiated state) also can bedetermined by analysis of morphological features of the stem cellculture. hESCs exhibit high nucleus to cytoplasm ratio with prominentnucleoli, and are rounded and typically grow in colonies that lietightly packed together. The borders of these colonies are very tight,rigid and well-defined. NSCs exhibit a flat, in some cases pavementedmorphology. NSCs tend to grow as clones, or in groups held very closelytogether, where they might take on a square or roughly triangularappearance (Temple, 1989; Thomson et al, 1998). Additionally, NSCs canbe put in culture as floating aggregates known as neurospheres. Afterone week, these neurospheres are dissociated into single cells andreplated in the same conditions. A properly maintained NSC line willcontinue to generate new spheres at the same rate or higher than theprevious passage. A deficit in NSC maintenance would result in reducedneurosphere formation after passage (Fasano et al, 2007).

VI. Kits

The sustained release compositions for culturing undifferentiated cells,such as stem and/or progenitor cells, described herein can be providedin a kit. The kit can include one or more sustained release compositionsof the invention (e.g., PLGA microspheres) containing one or more growthfactors suitable for maintaining such cells in an undifferentiatedstate; and, optionally, (b) informational material. Each sustainedrelease composition can be provided separately in the kit, if more thanone composition is included, or they can be provided as one or moremixtures of different sustained release compositions. In addition to theactive compound (e.g., growth factor), the sustained release compositionof the kit can include other ingredients, such as a solvent or buffer, astabilizer, a preservative, and/or an additional agent (e.g., growthfactor) for culturing and maintaining cells in an undifferentiatedstate, as described herein. The sustained release composition can beprovided in the kit as ready-to-use, i.e., containing all agents, e.g.,pH stabilizing agents (e.g., Mg(OH)₂), growth factor stabilizers (e.g.,heparin), and the active agent, e.g., growth factor(s), to be includedin the composition upon addition to a cell culture. Alternatively, thekit can provide some or each of the components of the final sustainedrelease composition separately, with instructions for how to combine thecomponents prior to use.

The informational material can be descriptive, instructional, marketingor other material that relates to the methods described herein and/or tothe use of the sustained release composition for the methods describedherein. For example, the informational material relates to the use ofthe sustained release composition provided in the kit for the culture ofundifferentiated cells (e.g., stem and/or progenitor cells). The kitscan also include paraphernalia for administering one or more compoundsto a cell (e.g., pipette, dropper, etc.).

The informational material of the kits is not limited in its form. Inmany cases, the informational material (e.g., instructions) is providedin printed matter, such as in a printed text, drawing, and/orphotograph, such as a label or printed sheet. However, the informationalmaterial can also be provided in other formats, such as Braille,computer readable material, video recording, or audio recording. Ofcourse, the informational material can also be provided in anycombination of formats.

The kit can include one or more containers for the sustained releasecomposition(s). In some embodiments, the kit contains separatecontainers, dividers or compartments for the composition andinformational material. For example, the composition can be contained ina bottle, tube or vial, and the informational material can be containedin a plastic sleeve or packet. In other embodiments, the separateelements of the kit are contained within a single, undivided container.For example, the sustained release composition is contained in a bottle,tube or vial that has attached thereto the informational material in theform of a label.

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

EXAMPLES Example 1 FGF2 Fluctuation In Standard hESC Culture Conditions

This Example demonstrates that FGF2 concentrations fluctuatedramatically in standard hESC culture conditions.

WA-09 hESCs (WiCell Research Institute, National Stem Cell Bank,Madison, Wis.) were plated on tissue culture treated dishes or plates instandard mouse embryonic fibroblast (MEF) feeder (Global Stem Inc.(Rockville, Md.)) or non-MEF (Matrigel) (BD Biosciences, Bedford, Mass.)conditions in complete medium containing 10 ng/ml FGF2 or inMEF-conditioned medium (MEF CM) containing 10 ng/ml FGF2. Completeculture medium contained DMEM/F12 (Invitrogen, Carlsbad, Calif.),containing L-Glutamine (Invitrogen), Minimal Essential Amino Acids(Invitrogen), 20% Knockout Serum Replacement (Invitrogen), and2-Mercaptoethanol (Invitrogen), and MEF CM was the same complete culturemedium, but prior to use had been allowed to bathe MEFs for 24 hours.

The media was changed daily according to standard hESC culture technique(see, Fasano et al., 2010). Before each re-feed (change of media), asample of media was collected and the levels of FGF2 were quantifiedusing a cytokine bead kit and FACS according to the manufacturerprotocols (BD Cat# 558327). After 5 hours of culture, in both MEF andMatrigel conditions, FGF2 levels were already reduced by greater than50%, and by the time of the next media change (24 hours later), FGF2levels were not detectable (FIG. 1A and FIG. 1B). These resultsdemonstrated that the standard method for maintaining hESCs in cultureis sub-optimal due to growth factor instability.

Example 2 FGF2-Containing Microspheres Sustain FGF2 Levels In hESCCultures

WA-09 hESCs (WiCell Research Institute,) were plated on Day -1 onstandard MEF (Global Stem Inc.) on tissue culture treated culture dishesor plates in standard hESC media containing DMEM/F12 (Invitrogen,Carlsbad, Calif.), containing L-Glutamine (Invitrogen), MinimalEssential Amino Acids (Invitrogen), 20% Knockout Serum Replacement(Invitrogen), and 2-Mercaptoethanol (Invitrogen) and 10 ng/ml FGF2.Biodegradable PLGA microspheres were produced using 0.5% FGF2 (R&D®Systems), 3% Mg(OH)₂, 1:1 weight ratio of heparin to FGF2 and 1 mM EDTA(“FGF2-microspheres”).

To measure FGF2 concentration in the hESC cultures over time, on Day 0,the culture media in the plated hESCs was changed and replaced withfresh complete media containing 10 ng/ml FGF2. Immediately after themedia was replaced, a sample was collected in order to determine theconcentration of FGF2 on Day 0 (the starting concentration). Just afterthe media sample was collected, FGF2-containing microspheres were addedto one group (“FGF2-microspheres”). 7 μl per 1 ml of culture media ofFGF2-microsphere preparation (concentration 1000 microspheres per 1 μlof preparation) were used. Six (6) hours later, and then every 24 hours(calculated from the time of media replacement on Day 0), a sample ofmedium was collected and the level of FGF2 in each culture wasquantified using a cytokine bead kit and FACS according to manufacturerprotocols (BD Cat #558327). Consistent with Example 1, above, FGF2levels in the soluble FGF2 cultures declined rapidly, and FGF2 could notbe detected after 1 day of culture. However, in the FGF2-microsphereculture, FGF2 levels were stable, with only 20% of the starting level ofFGF2 being reduced by Day 3 (FIG. 2). These data demonstrated thatsustained release compositions, such as PLGA microspheres, can be usedto maintain stable growth factor levels in cell culture, therebyoffering a solution to a current problem with the standard methodologyfor stem cell culture; namely rapid FGF2 level fluctuation and decline.

Example 3 Culture of hESCs With FGF2-Containing PLGA Microspheres

On Day −1, WA-09 hESCs were plated on standard mouse embryonicfibroblast (MEF) and non-MEF conditions as described in Example 1,above, in complete culture media containing DMEM/F12 (Invitrogen,Carlsbad, Calif.), containing L-Glutamine (Invitrogen), MinimalEssential Amino Acids (Invitrogen), 20% Knockout Serum Replacement(Invitrogen), 2-Mercaptoethanol (Invitrogen) and 10 ng/ml FGF2. The cellculture media was not changed for the 5-day duration of the experiment.

Biodegradable PLGA microspheres containing 0.5% soluble human FGF2 (R&DSystems), 3% Mg(OH)₂, 1:1 weight ratio of heparin to FGF2 and 1 mM EDTA(“FGF2 microspheres”) or control microspheres containing 3% Mg(OH)₂, 1:1weight ratio of heparin to FGF2 and 1 mM EDTA, but not FGF2 (“emptymicrospheres”), were added to some of the cell cultures on Day 0 (24hours after plating), following replacement of the culture media withcomplete hESC culture media supplemented with 10 ng/ml FGF2, eitherdirectly to the well containing hESCs or in a transwell above hESCs. 7μl per 1 ml of culture media of FGF2-microsphere preparation(concentration 1000 microspheres per 1 μl of preparation) were used. Ina control group, 10 ng/ml soluble human FGF2 was added to the culturedaily (“soluble FGF2”).

After 5 days, cells were assessed for SSEA-4 expression via FACS. TheSSEA-4-antibody used for FACS staining was obtained from BD Biosciences.As expected, cells cultured under the empty microsphere conditionexhibited less SSEA-4 expression after five days compared to cells thatreceived daily soluble FGF2 (FIG. 3). However, cells cultured withFGF2-microspheres exhibited a slight increase of SSEA-4, indicating thatthese cells underwent even less spontaneous differentiation than withdaily soluble FGF2 treatment (FIG. 3). Further, undifferentiatedmorphology of the cells was observed in cells treated with soluble FGF2or FGF2 microspheres, and differentiated morphology was observed in thenegative control (empty microspheres).

In addition to FACS analysis, mRNA expression levels of OCT-4, anundifferentiated hESC maker, and the differentiation markers Brachyuryand Sox17 were determined by quantitative RT-PCR. Verified TaqMan probesfrom Applied BioSystems™ (Foster City, Calif.) were used to assess geneexpression. In culture, hESCs typically undergo low levels ofspontaneous differentiation using standard stem cell culture methods(i.e., daily addition of soluble FGF2). Surprisingly, compared to dailysoluble FGF2 treatment, hESCs cultured in the presence of the stableconcentration range of FGF2 provided by the PLGA microspheres exhibitedsignificantly less spontaneous differentiation, as indicated bydecreased mRNA expression levels of Brachyury and Sox17 and higher OCT-4mRNA expression levels (FIG. 4). Similar results were obtained when FGF2microspheres were placed in transwells above hESCs, indicating thatdirect contact of the microspheres with the stem cells was not required.Results from the mRNA expression analysis of OCT-4, Brachyury and SOX17were consistent with morphological culture homogeneity.

Thus, the culture methods used in this Example resulted in themaintenance of a stable FGF2 concentration range in the hESC cultures,and in the surprising decrease in spontaneous differentiation of hESCs,a problem associated with standard culture methods (i.e., daily, directaddition of soluble FGF2 to the culture media). The methods described inthis Example therefore provide the benefit of increasing the utility ofcultured hESCs and significantly reducing the labor required to growthem.

Example 4 Sustained Release of FGF2 In Long-Term hESC Cultures

WA-09 hESCs were plated on standard MEF and cultured in complete hESCculture media containing 10 ng/ml FGF2, as described in Example 1,above. Biodegradable PLGA microspheres were produced using 0.5% FGF2(R&D® Systems), 3% Mg(OH)₂, 1:1 weight ratio of heparin to FGF2 and 1 mMEDTA (“FGF2-microspheres”). 7 μl per 1 ml of culture media ofFGF2-microsphere preparation (concentration 1000 microspheres per 1 μlof preparation) were used. Cells were either fed every day, every 3rdday, or once with 10 ng/ml soluble FGF2, or cells were fed every 3rd daywith FGF2-microspheres or once with FGF2-microspheres. After 7 days,hESCs were passaged according to standard procedure [Fasano et al.,2010, supra], and some cells were analyzed by FACS to assess theexpression of the pluripotency markers, SSEA-4 and SSEA-3, and thedifferentiation marker SSEA-1. Cells were passaged every 7 days, for 5weeks. After 35 days, the percentage of cells that stained positive forSSEA-1, SSEA-3, and SSEA-4 in each group was determined. The expressiondata was similar for cells fed once with FGF2 microspheres or every3^(rd) day. Cells that were fed FGF2-microspheres maintained highexpression of pluripotency markers SSEA-3 and SSEA-4 and low expressionof differentaion marker SSSEA-1, indicating that the cells weremaintained in an undifferentiated state in the long-term culture (FIG.5A).

In addition to this, at Day 14 and Day 21, RNA was isolated from thecultured cells and mRNA expression of the pluripotency marker NANOG wasquantified by qRT-PCR. Surprisingly, at both time points, there werehigher levels of NANOG expression in all groups of cells cultured withFGF2 microspheres compared to cells fed every day with soluble FGF2.These results thus showed that cells cultured long-term in the presenceof FGF2 microspheres had consistent pluripotent marker expression, and,in some assays, higher pluripotent marker expression levels than cellscultured using standard culture methods (FIG. 5B). The stable FGF2levels provided by a sustained release composition, such as FGF2microspheres, can therefore minimize the number of culture feeds andmaintain hESCs in a more undifferentiated state.

Example 5 Sustained Range of FGF2 Concentration Maintains hESCs In AMore Undifferentiated State Without Feeder Cells Or Feeder ConditionedMedium

hESCs can be grown without MEF feeders only in the presence of MEFconditioned hESC media, or, in expensive, “special” media containingvery high levels of FGF2. In this experiment, it was determined whethersustained FGF2 levels provided by FGF2 microspheres was sufficient forthe culture of hESCs in the absence of MEF feeders, MEF conditioned hESCmedia, or special media. WA-09 hESCs were grown on Matrigel in hESCculture media, as described in Example 1. PLGA microspheres, containing0.5% FGF2, 3% Mg(OH)₂, 1:1 weight ratio of heparin to FGF2 and 1 mM EDTA(“FGF2 microspheres”) were added to some cell culture wells to provide asustained FGF2 level. 7 μl per 1 ml of culture media of FGF2-microspherepreparation (concentration 1000 microspheres per 1 μl of preparation)were used. In the control group (treated using the “standard method”),soluble human FGF2 was added every day at 10 ng/ml in fresh media at thetime of media change. In other groups, FGF2-microspheres were added tothe medium either once or twice a week.

The cells were grown for 5 days, and then mRNA expression levels of thepluripotency marker OCT-4 and the differentiation marker SOX17 weredetermined by quantitative RT-PCR. Cells cultured in the presence ofFGF2 microspheres expressed higher OCT-4 levels than cells treated dailywith soluble FGF2 (FIG. 6). Surprisingly, in all groups cultured in thepresence of FGF2 microspheres, mRNA expression of the differentiationmarker SOX17 was undetectable (FIG. 6). These data demonstrated that thestable, sustained FGF2 levels provided by a sustained releasecomposition, such as FGF2 microspheres, can maintain hESCs in a moreundifferentiated state without MEF feeder cells, MEF feeder conditionedmedium, or specialized culture medium, as described above.

Example 6 Sustained FGF2 Levels Improved the Pluripotency of ADifferentiated hESC Culture

If cultured in media without FGF2, hESCs will slowly start todifferentiate, gradually becoming less pluripotent. In this Example, itwas tested whether sustained FGF2 levels could restore hESC pluripotencyand make the culture more homogeneously pluripotent, in hESCs that hadbeen allowed to differentiate.

hESCs were fed upon plating with complete culture medium, as describedin Example 1, containing 10 ng/ml soluble FGF2 and then either fedsoluble FGF2 every day (“FGF2 Daily”) or not fed again (“No FGF2”), andleft in culture for 7 days. On Day 7, cells were passaged according tostandard procedures [see, Fasano et al., 2010, supra], and some cellswere assessed for protein expression of the pluripotency markers SSEA-3and SSEA-4. Expression of both SSEA-3 and SSEA-4, as determined by FACSanalysis, was found to be decreased in the cells that did not receivedaily FGF2 (“no FGF2” group) compared to expression of those markers incells fed daily FGF2 (“FGF2 daily”) (FIG. 7A).

At this point, the cells in both groups were re-plated, and regularFGF2-containing complete media or complete media containingFGF2-microspheres (containing 0.5% FGF2, 3% Mg(OH)₂, 1:1 weight ratio ofheparin to FGF2 and 1 mM EDTA) were added to the cultures. 7 μl per 1 mlof culture media of FGF2-microsphere preparation (concentration 1000microspheres per 1 μl of preparation) were used. Cells were fed everyday with 10 ng/ml FGF2 (“Every Day” condition), or once (on Day 0) ortwice (on Days 0 and 2) with FGF2-microspheres (“Beads Once” or “BeadsTwice per Week” conditions, respectively). After 7 additional days ofculture following re-plating, expression levels of SSEA3 were once againdetermined. SSEA-3 expression levels were higher in all groups culturedin the presence of FGF2-microspheres (“Beads Once” or “Beads Twice perWeek” conditions), compared to cells fed daily with soluble FGF2 (“EveryDay” condition). In the “no FGF2” group, every day feeding of solubleFGF2 following re-plating did not restore SSEA-3 back to control levels(i.e., the percentage of SSEA-3+cells in the FGF2 Daily group fed everyday with soluble FGF2 during the 7 additional days of culture), however,in the group of cells fed once with FGF2 microspheres, pluripotency(expression of SSEA-3) was partially rescued, and in the group fedFGF2-microspheres twice per week, pluripotency (expression of SSEA-3) ofdifferentiated cells was nearly completely rescued, as compared to thecontrol levels (FIG. 7B).

Example 7 Culture of NSCs With FGF2 PLGA Microspheres

Mouse NSCs were isolated from embryonic cortical brains and placed inculture and maintained as previously described [Qian et al, 1997, supra]or with modifications to that protocol, as described below. Briefly, insome wells, cells were plated in a basal medium supplemented with 10ng/ml FGF2 and the media was changed every third day. This was the“standard condition.” In other wells, NSCs were cultured in basic mediumwithout soluble FGF2 in the presence of empty microspheres alone (“noFGF2”, as in Example 3) to show that the microspheres alone are notmitogenic. In other wells, cells in basic medium without soluble FGF2were cultured in the presence of PLGA microspheres containing 0.5% FGF2,3% Mg(OH)₂, 1:1 weight ratio of heparin to FGF2 and 1 mM EDTA, andnothing more was added to these wells during culture (the culture mediawas not changed, and nothing was added to the culture after addition ofthe microspheres).

Following 7 days of treatment, cells were stained with Nestin—(DSHB) andβ-tubulin—(Sigma-Aldrich) specific antibodies using standard stainingtechniques [see, Fasano et al., 2007, supra; and Fasano et al., 2009,Genes Dev. (2009) Mar 1; 23 (5):561-74]. In the standard condition, cellclones generated as expected with a mix of NSCs and NPCs and neurons(differentiated cells) indicated by positive staining for Nestin (forNSCs and NPCs) or β-Tubulin (neurons (differentiated cells)).Specifically, cells fed with soluble FGF2 every third day gave rise tomixed clones with 380 NSCs and NPCs (Nestin+) and 170 neurons(β-tubulin+) (FIG. 8). Wells with the empty microspheres had less totalcells, indicating poor survival with very little Nestin staining andloss of NSCs and NPCs (FIG. 8). In contrast, wells that had FGF2containing microspheres (“FGF2 microspheres”) added directly to themexhibited more clonal growth, with dramatically higher numbers ofNestin+ cells, indicating better NSC and NPCs growth. Specifically, onetreatment with FGF2 containing microspheres increased cell numberdramatically as well as the proportion of NSCs and NPCs: 1440 (Nestin+)to 120 neurons (β-tubulin) (FIG. 8).

Using standard (i.e., conventional) stem cell culture methods, the slow,steady differentiation of NSCs limits their usefulness. However, thenovel methods discovered and described herein, in which a stable FGF2concentration range was maintained over time in the stem cell cultureusing PLGA microspheres, resulted in the surprising decrease inspontaneous differentiation of cultured NSCs, thereby increasing theutility of these cells and significantly reducing the labor required togrow them. In one experiment, cells in additional wells were cultured inthe standard condition and additionally with empty microspheres todetermine whether the microspheres had any toxic effects on the cells.Addition of the empty microspheres to cells cultured under the standardcondition did not affect the cells (i.e., was not toxic) compared to thestandard condition. These data show that constant FGF2 exposure viasteady delivery of FGF2 by the PLGA microspheres maintains NSCs and NPCsin the undifferentiated state surprisingly better than standard culturemethods.

Example 8 Frequent Administration of FGF2 To NSCs

In this experiment, NSCs were fed every 8 hours with 10 ng/ml solublehuman FGF2, to achieve a constant supply of FGF2, over 6 days. NSCs andNPCs maintain the stem or progenitor cell fate (i.e., remain in anundifferentiated state) by cell to cell interactions; loss of thiscontact initiates a differentiation response. Thus, tighter colonies areindicative of less differentiated NSCs and NPCs. Following frequentfeeding (every 8 hours) of NSCs for 6 days, the NSC culture had tighterlooking cells, indicative of undifferentiated cells. The NSC culturealso had more Nestin staining, and less differentiation as measured byTuj1 staining, compared to the standard method (feeding FGF2 every thirdday) (FIG. 9). This data demonstrated that a constant FGF2 supply (e.g.,by frequent feeding) can maintain NSC cultures in a less differentiatedstate better than the standard protocol.

Example 9 Culture of Human RPESCs In Presence of Sustained Levels ofFGF2

This example demonstrates the use of sustained FGF2 levels to propagatehuman retinal pigment epithelial stem cells (RPESCs) in culture. RPESCswere isolated as follows:

Dissection

Human eyes from 22-99 year old donors were obtained from The Eye-Bankfor Sight Restoration, Inc. (New York, N.Y.), and the National DiseaseResearch Interchange (NDRI) (Philadelphia, Pa.). The eyes were cut atthe ora serrata and the anterior segment discarded. The vitreous andretina were removed leaving the RPE layer exposed. RPE dissection andsingle cell dissociation was performed as previously described [see,U.S. Patent Application Publication No. 2009/0274667 by Temple; Burke,C. M. et al., Exp Eye Res 62, 63 (1996); and Maminishkis, A. et al.,Invest Ophthalmol Vis Sci 47, 3612 (2006)]. Gentle trituration withinthe eyecup using care to maintain Bruch's membrane yielded a suspensionof RPE cells with minimal contamination by rod outer segments, blood orother cell types.

RPE Sheet Dissection And Cobblestone Culture

After the vitreous and retina were separated as described above, theeyecup was rinsed with sterile PBS and then incubated with celldissociation buffer enzyme-free Hanks'-based (Gibco-Invitrogen,Carlsbad, Calif.) for 10 minutes at 37° C. Gently the dissociationbuffer was removed and the eyecup filled with DMEM/F12 mediasupplemented with 20% FBS (Gibco-Invitrogen). Using a dulled, angled,double bevel spoon blade (3.0 mm), small sheets (1 mm²) of RPE wereremoved from the Bruch's membrane by gentle scraping. RPE sheets wereplated into Matrigel (BD Biosciences) pre-treated tissue culture platesand cultured in RPE medium: MEM-α modified medium (Sigma-Aldrich), 2 mML-glutamine, penicillin/streptomycin (1:100), 1% Na-Pyruvate, 10% FBS(fetal bovine serum), supplemented with THT (Taurine Hydrocortisone,Triiodo-thyronin) and N1 (Sigma-Aldrich) as described [De, S. et al.Arch Ophthalmol. 2007; May; 125 (5):641-5; Burke et al., 1996, supra;and Maminishkis, A. et al., 2006, supra] with 10 ng/ml FGF2 and 1 ng/mlEGF (Gibco-Invitrogen).

Culture

Using a defined medium growth medium containing MEM-α modified medium(Sigma-Aldrich), 2 mM L-glutamine, penicillin/streptomycin (1:100), 1%Na-Pyruvate, 10% FBS (fetal bovine serum), supplemented with THT(Taurine Hydrocortisone, Triiodo-thyronin) and N1 (Sigma-Aldrich) andsupplemented with 7 μl per 1 ml of culture media of FGF2-containing PLGAmicrospheres (containing 0.5% FGF2, 3% Mg(OH)₂, 1:1 weight ratio ofheparin to FGF2 and 1 mM EDTA), the RPESCs were grown for 5 days. Cellswere grown according to the methods described in detail in U.S. PatentApplication Publication No. 2009/0274667 by Temple et al. After 5 days,the cells were photographed and displayed normal morphology and growthpatterns compared to medium with soluble FGF2 (FIG. 10).

Example 10 Generation of iPSCs In the Presence of FGF2

This example demonstrates the use of sustained FGF2 levels for thegeneration of induced pluripotent stem cells (iPSCs). Adult fibroblastswere infected with retroviral supernatant containing OCT4, KLF4, c-MYCand SOX2, according to the methods described in Takahashi, K. et al.(2007); Cell 131, 861-872. 10 days after infection, cells weretrypsinized and counted and 10⁵ cells were plated onto MEFs in DMEM+10%FBS. The day after, media was replaced with standard hESC media(DMEM/F12 (Invitrogen, Carlsbad, Calif.)), containing L-Glutamine(Invitrogen), Minimal Essential Amino Acids (Invitrogen), 20% KnockoutSerum Replacement (Invitrogen), and 2-Mercaptoethanol (Invitrogen))+4ng/ml FGF2 (“soluble FGF2” group) or with hESC media containingFGF2-containing PLGA microspheres (containing 0.5% FGF2, 3% Mg(OH)₂, 1:1weight ratio of heparin to FGF2 and 1 mM EDTA) (“FGF2-microspheres”group). In the soluble FGF2 group, the media was replaced daily, whilemedia containing FGF2-containing PLGA microspheres was changed twice perweek (the fresh media added to the cultures at the time of media changecontained FGF2-microspheres). Pictures were taken 9 days after cellswere plated. In the condition with daily feeds and soluble FGF2, cellswere observed to have no distinct colony size or formation (FIG. 11,left panel). In contrast, cells cultured in the presence ofFGF2-microspheres had colonies that exhibited characteristics of humanembryonic stem cell colonies (FIG. 11, right panel), demonstrating thatculture of iPSCs in the presence of sustained levels of FGF2 increasedthe efficiency of iPSC generation.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are incorporated by reference in theirentirety as if physically present in this specification and to the sameextent as if each item had been incorporated by reference individually.However, the citation of any such material, even in discussing theBackground of this invention, is not to be construed as an admissionthat the material was or is available as prior art to the presentinvention.

1. A method for culturing a mammalian stem or progenitor cell, whereinthe method comprises incubating the stem or progenitor cell in thepresence of a sustained release composition containing at least onegrowth factor, wherein the sustained release composition releases thegrowth factor, and wherein the presence of the growth factor maintainsthe cell in an undifferentiated state.
 2. The method of claim 1, whereinthe sustained release of the growth factor maintains the concentrationof the growth factor in the cell culture in a stable concentration rangeof 80%-100% of the starting concentration of growth factor.
 3. Themethod of claim 1, wherein the sustained release of the growth factormaintains the concentration of the growth factor in the cell culture ina stable concentration range of 80%-95% of the starting concentration ofgrowth factor.
 4. The method of claim 1, wherein the sustained releaseof the growth factor maintains the concentration of the growth factor inthe cell culture in a stable concentration range of 80%-90% of thestarting concentration of growth factor.
 5. The method of claim 1,wherein the sustained release composition releases at least one growthfactor over a period of at least about 1 day.
 6. The method of claim 1,wherein the sustained release composition is apoly(DL-lactide-co-glycolide) (PLGA) microsphere.
 7. The method of claim6, wherein the concentration of the PLGA microsphere ranges from about 5to about 300 ng/ml.
 8. The method of claim 1, wherein the sustainedrelease composition further comprises heparin or dextran sulfate.
 9. Themethod of claim 1, wherein the sustained release composition furthercomprises Mg(OH)₂.
 10. The method of claim 1, wherein the cell ismaintained in an undifferentiated state for at least about 3 days. 11.The method of claim 1, wherein the stem cell is a cell selected from thegroup consisting of an embryonic stem cell (ESC), an induced pluripotentstem cell (iPSC), a neural stem cell (NSC), a retinal pigment epithelialstem cell (RPESC), a hematopoietic stem cell (HSC), a mesenchymal stemcell (MSC), a cancer stem cell (CSC), and an epiblast stem cell.
 12. Themethod of claim 1, wherein the stem cell is a human embryonic stem cell.13. The method of claim 1, wherein the progenitor cell is a neuralprogenitor cell.
 14. A method for culturing a stem or progenitor cell,wherein the method comprises incubating the stem or progenitor cell inthe presence of a sustained release composition comprising fibroblastgrowth factor 2 (FGF2), wherein the sustained release compositionreleases FGF2.
 15. The method of claim 14, wherein the stem orprogenitor cell is maintained in an undifferentiated state for at leastabout 3 days.
 16. The method of claim 14, wherein the sustained releasecomposition releases the FGF2 over a period of at least 1 day.
 17. Themethod of claim 14, wherein the sustained release composition comprisesFGF2 at a concentration of 0.5% (w/v) at the start of incubation withthe stem or progenitor cell.
 18. The method of claim 14, wherein thesustained release of FGF2 maintains the concentration of FGF2 in thecell culture in a stable concentration range of 80%-100% of the startingconcentration of FGF2.
 19. The method of claim 14, wherein the sustainedrelease of FGF2 maintains the concentration of FGF2 in the cell culturein a stable concentration range of 80%-95% of the starting concentrationof FGF2.
 20. The method of claim 14, wherein the sustained release ofFGF2 maintains the concentration of FGF2 in the cell culture in a stableconcentration range of 80%-90% of the starting concentration of FGF2.21. The method of claim 14, wherein the sustained release compositionfurther comprises one or more of the agents selected from the groupconsisting of heparin, dextran sulfate, a polyanion that complexes withFGF2, Mg(OH)₂, and EDTA.
 22. The method of claim 14, wherein thesustained release composition is a poly(DL-lactide-co-glycolide) (PLGA)microsphere.
 23. The method of claim 16, wherein the concentration ofthe PLGA microsphere ranges from about 5 ng/ml to about 300 ng/ml. 24.The method of claim 14, wherein the sustained release compositionfurther comprises 1.0% (w/v) heparin or 1.0% (w/v) dextran sulfate. 25.The method of claim 24, wherein the ratio of heparin or dextran sulfateto FGF2 is about 2:1.
 26. The method of claim 14, wherein the sustainedrelease composition further comprises 3% (w/v) Mg(OH)₂.
 27. The methodof claim 14, wherein the sustained release composition further comprises1 mM EDTA.
 28. The method of claim 14, wherein the stem cell is selectedfrom the group consisting of an embryonic stem cell, a inducedpluripotent stem cell, a neural stem cell, a retinal pigment epithelialstem cell, a hematopoetic stem cell, a mesenchymal stem cell, a cancerstem cell and an epiblast stem cell.
 29. The method of claim 14, whereinthe stem cell is a human embryonic stem cell.
 30. The method of claim14, wherein the progenitor cell is a neural progenitor cell.
 31. Themethod of claim 14, wherein the sustained release composition furthercomprises one or more additional growth factors.
 32. The method of claim14, wherein the one or more additional growth factors are selected fromthe group consisting of epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), sonic hedgehog (Shh), leukemia inhibitory factor(LIF) and a Wnt protein.
 33. A method for culturing an undifferentiatedmammalian cell, wherein the method comprises incubating theundifferentiated mammalian cell in the presence of a sustained releasecomposition containing at least one growth factor, wherein the sustainedrelease composition releases the growth factor, wherein the releasedgrowth factor is maintained within a predetermined concentration range,and wherein the maintenance of the released growth factor within saidconcentration range maintains the cell in an undifferentiated state. 34.A method for culturing a mammalian stem or progenitor cell, wherein themethod comprises incubating the stem or progenitor cell in the presenceof a stable concentration range of at least one growth factor over aperiod of at least 1 day.
 35. The method of claim 34, wherein the stableconcentration range of growth factor is maintained by continuous releaseof the growth factor using mechanical means.
 36. The method of claim 34,wherein the stable concentration range of growth factor is maintained bycontinuous release of the growth factor by a sustained releasecomposition.